MiR-199a-5p-HIF-1α-STAT3 positive feed-back loop contributes to the progression of NSCLC

Non-small Expression of miR-199a-5p and HIF-1α mRNA were quantied employing qRT-PCR. H1299 and A549 cells were transiently transfected with miR-199a-5p mimics or inhibitors. Then CCK-8 assays, ow cytometry analysis, Transwell assay were implemented for detecting cell proliferation, cell cycle, apoptosis, migration and invasion of NSCLC cells, respectively. HIF-1α, STAT3 and p-STAT3 expression were detected via Western blotting. Bioinformatic analysis and dual-luciferase assay were performed for monitoring interaction between miR-199a-5p, HIF-1α and STAT3. Xenograft models were established with nude mice for further analyzing Bevacizumab resistance of NSCLC. progression of NSCLC.


Introduction
Lung cancer, is the most common and leading cause of cancer-related death worldwide, among which, non-small cell lung cancer (NSCLC), accounts for more than 80% of cases [1]. Even though novel therapeutic approaches for NSCLC have signi cantly improved the the prognosis of the patients, almost all of the current approved drugs face the problem of drug resistance [2][3][4]. It's still necessary to further clarify the mechanism of sustaining progression of NSCLC, which may provide clues for developing novel therapy strategies to improve the survival time of patients.
Non-coding RNAs, including microRNAs (miRNAs), participate in the pathogenesis of diverse human diseases, inclusive of NSCLC [5]. MiR-199a-5p, markedly downregulated in NSCLC tissues, is con rmed as a tumor suppressor: via directly targeting MAP3K11, miR-199a-5p suppresses proliferation and induces cell cycle arrest of A549 and H1299 cells [6]. Another research pointed out that overexpression of miR-199a-5p is capable of increasing doxorubicin sensitivity of A549 and H460 cells [7]. However, the explicit function and underlying mechanism whereby miR-199a-5p regulates NSCLC progression remain largely unknown.
Hypoxia inducible factor-1 (HIF-1α), de ned as an oxygen-regulated protein, is in marked association with cancer biology [8]. Of note, in NSCLC, HIF-1α was substantiated to be an oncogene. For example, subsequent to knockdown of HIF-1α with siRNA, proliferation and invasion of NCI-H157 cells were constrained but apoptosis was promoted [9]. Signal transducer and activator of transcription 3(STAT3) is also identi ed as a promoter for NSCLC. For instance, downregulation of STAT3 weakens colony forming ability and proliferation of A549 and SK-MES-1 cells [10]. Nonetheless, the mechanism of HIF-1α and STAT3 hyperactivation in NSCLC has not been clari ed clearly.

Cell counting kit-8 (CCK-8) assay
A549 and H1299 cells were seeded into 96-well plates (2 × 10 3 per well). Followed by culture for 24,48,72 and 96 h, each well was supplemented with 10 µL CCK-8 solution (Dojindo, Tokyo, Japan). After CCK-8 solution was added, and the cells were incubated for 1 h. Then cell viability at each time point was evaluated by measuring the absorbance values at a wavelength of 450 nm. Then the proliferation curve was plotted.

Flow cytometery analysis
For cell cycle analysis,A549 or H1299 cells were seeded into 6-well plate (5 × 10 5 cells / well) and cultured in 0.2% serum medium for 24 h for synchronization. Subsequently, the cells were stained with propidium iodide (PI) solution (Beyotime, Shanghai, China) and were examined employing a FACS ow cytometer (BD Biosciences, San Jose, CA, USA). For apoptosis analysis A549 and H1299 cells were collected, washed, xed, and permeabilized. Then Annexin V-FTIC/PI Apoptosis Detection Kit (KeyGen, Nanjing, China) was employed to detect the apoptosis of the cells according to the manufacturer's instruction. The data were computationally analyzed deploying ModFit software (BD Biosciences, San Jose, CA, USA) (for cell cycle analysis) or Flowjo V10 software (BD Biosciences, San Jose, CA, USA) (for apoptosis analysis).

Transwell assay
The pore size of the aperture in the membrane of Transwell chambers (Corning, Shanghai, China) was 8 µm. The chambers were coated with Matrigel (Sigma, Shanghai, China) for determination of invasive ability. No matrigel was added in the migration assay. The lower compartment of the Transwell system was lled with 600 µL RPMI-1640 medium containing 10% FBS, and the upper compartment contained 200 µL cell suspension (5 × 10 5 cells in each well in serum-free medium). The cells were cultivated for 48 h, and then the Transwell chambers were taken out. The cells that failed to migrate were removed with cotton swabs. Then the remaining cells attached on the lower surface of the Transwell membrane were xed with 4% paraformaldehyde for 20 min, and stained with 0.5% crystal violet solution. Ultimately, 5 random elds of each Transwell membrane were randomly selected and the numbers of stained cells were reckoned under a microscope.

Dual-luciferase reporter assay
The fragment of wild type (WT) or mutant (MUT) HIF1A 3′ UTR was inserted into a pmirGLO -dualluciferase miRNA target expression vector (Promega, Madison, WI, USA). The recombinant reporter vectors were co-transfected into H1299 cells with miR-199a-5p mimics or mimics control and then the cells were cultivated for 48 h. Luciferase activity was subsequently measured utilizing Dual Luciferase Reporter Gene Assay Kit (Beyotime, Jiangsu, China) in conformity with manufacturer's protocol. The luciferase activity of re y was normalized to that of of renilla.

Western blotting
NSCLC cells in each group were harvested and washed three times with cold PBS, and 100 µL RIPA lysate (Beyotime, Shanghai, China) was added. The cells were lysed in ice-bath for 30 min to extract the total protein. The mixtures were succeedingly centrifuged at 12,000 rpm for 20 min, and the supernatant was collected as the protein samples. The protein samples were denatured, separated by sodium dodecylsulfate-polyacrylamide gel-electrophoresis, and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% skimmed milk, and incubated with primary antibodies [Rabbit Anti-STAT3 (phospho S727) antibody, 1:1000, ab32143; Rabbit Anti-STAT3 antibody, 1:1000, ab68153; Rabbit Anti-HIF-1 alpha antibody, 1:1000, ab82832] at 4 °C overnight.
Afterward, the membranes were washed with TBST buffer and then incubated with secondary horseradish peroxidase (HRP-) labeled goat anti-rabbit IgG antibodies (1:2000, ab205718, Abcam, Shanghai, China) for additional 2 h at room temperature. β-actin was employed as internal reference. The protein bands on the membranes were then detected utilizing ECL kit (Amersham Pharmacia Biotech, Little Chalfont, UK) and computationally analyzed by deploying Image J software (National Institutes of Health, Bethesda, MD, USA).

Xenografts models
The procedures of animal experiments have been reviewed and approved by the Animal Ethics Committee of the Sixth A liated Hospital of Sun Yat-sen University and in compliance with the National Institute of Health Guidelines for Care and Use of Laboratory Animals in Biomedical Research. A549 cells, previously reported to be resistant to Bevacizumab, were selected for establishing xenografts models.
Subsequent to transfection with miR-199a-5p mimics, miR-199a-5p inhibitors, HIF-1α and STAT3 overexpression plasmid or their negative controls, A549 cells were inoculated subcutaneously into the right ank region of male athymic (BALB/c-nu) mice (6 weeks old). By the time of tumor size reached approximately 100 mm 3 , 27 mice were randomly divided into nine groups with 3 mice per group: Blank group (no treatment), BV group (mice were injected with 10 mg/kg Bevacizumab weekly), BV + control of mimics group (A549 cells were transfected with control of mimics prior to injection and mice were injected with 10 mg/kg Bevacizumab weekly. As for the rest groups, A549 cells were transiently transfected with different plasmids or oligonucleotides prior to injection and mice were injected with 10 mg/kg Bevacizumab weekly: BV + control of inhibitors group, BV + miR-199a-5p mimics group, BV + miR-199a-5p inhibitors group, BV + miR-199a-5p mimics + vector group, BV + miR-199a-5p mimics + HIF-1α group, BV + miR-199a-5p mimics + STAT3 group. The tumor volume was recorded every two days. The tumor volume (in mm 3 ) was calculated with the formula 0.5 × L × W2 (L = length, W = width).

Statistical analysis
All statistical analyses were carried out utilizing SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). Data were presented as the means ± SE. Data were examined whether they were normally distributed with the One Sample Kolmogorov-Smirnov test. As for the data normally distributed, t-test or one-way ANOVA test was performed. As for the data not normally distributed, rank test was conducted. P < 0.05 was deemed to be statistically signi cant.

Expression of miR-199a-5p was down-regulated in NSCLC
GSE135918 contained the miRNA expression pro le of 5 fresh lung cancer tissues and 5 fresh non-tumor lung tissues. It was reanalyzed, and it was noted that miR-199a-5p was dramatically downregulated in NSCLC tissues versus normal lung tissues ( Fig. 2A, B). Consistently, qRT-PCR data manifested that miR-199a-5p was notably reduced in NSCLC tissues as opposed to adjacent normal lung tissues (Fig. 3A). Additionally, miR-199a-5p was observed to be reduced in serum samples of NSCLC patients versus healthy subjects (Fig. 3B). The downregulation of miR-199a-5p in NSCLC was further validated in GSE53882 and ENCORI database (Fig. 3C, D). Furthermore, miR-199a-5p expression was demonstrated to be underexpressed in NSCLC cell lines compared with normal lung epithelial cells BASE-2B (Fig. 3E).
What's more, the apoptosis of H1299 and A549 cells was analyzed and it was noted that miR-199a-5p overexpression promoted the apoptosis of NSCLC cells (Fig. 4E). Additionally, Transwell assays suggested that miR-199a-5p constrained the migration and invasion of NSCLC cells, while inhibiting miR-199a-5p promoted the migration and invasion of NSCLC cells (Fig. 4F, G). Collectively, these data validated that miR-199a-5p was tumor suppressor of NSCLC.
3.3 HIF-1α was con rmed as a target gene of miR-199a-5p miRmap, miRanda and TargetScan database were searched for the downstream target genes of miR-199a-5p and 221 genes were predicted by all of the three database (Fig. 5A). HIF1A, the gene of HIF-1α, previously reported to exert a tumor promoting role in NSCLC [9,13], was among them (Fig. 5B). Dualluciferase reporter assay was employed for verifying the binding site, and as shown, miR-199a-5p mimics remarkably suppressed luciferase activity of wild type HIF1A reporter, whereas this effect was abolished when the binding site was mutated (Fig. 5C). Furthermore, via qRT-PCR and Western blotting, it was demonstrated miR-199a-5p is capable of repressing the expressions of HIF-1α mRNA and protein, while inhibiting miR-199a-5p promoted the expression of HIF-1α in NSCLC cells (Fig. 5D, E). Besides, HIF-1α expression in NSCLC cell lines and tissues was signi cantly up-regulated (Fig. 5F, G). Moreover, the expression of miR-199a-5p in NSCLC tissues was in negative correlation with HIF1A (Fig. 5H). These data con rmed the targeting relationship between miR-199a-5p and HIF1A.

MiR-199a-5p enhanced sensitivity of A549 cells to Bevacizumab
High expression of HIF-1α and the activation of STAT3 signaling are reported to be conducive to resistance to Bevacizumab [14,15]. Therefore, we supposed that miR-199a-5p could be a potential therapeutic target for sensitizing NSCLC cells to Bevacizumab treatment. A549 cells, previously reported to show moderate resistance to Bevacizumab treatment [16], were used for subsequent experiments. MiR-199a-5p mimics or inhibitors were transiently transfected into A549 cells, and then the cells were treated with Bevacizumab (10 µM). Next, the expressions of HIF-1α, STAT3 and p-STAT3 were measured utilizing qRT-PCR, the results of which suggested that miR-199a-5p inhibited the expression of HIF-1α and activation of STAT3 (Fig. 8A-E). Through CCK-8 assay and ow cytometry, miR-199a-5p mimics were demonstrated to remarkably inhibit the viability and promote the apoptosis induced by Bevacizumab; conversely, miR-199a-5p inhibitors repressed the viability of A549 cells, and promoted the apoptosis induced by Bevacizumab (Fig. 8F-H). Consistently, the results of in vivo experiments suggested that miR-199a-5p facilitated the tumor-suppressive effects of Bevacizumab, while miR-199a-5p counteracted the tumor cytotoxicity of Bevacizumab (Fig. 8I, J). Collectively, these ndings indicated that miR-199a-5p enhanced sensitivity of A549 cells to Bevacizumab.

HIF-1α and STAT3 enhanced resistance of A549 cells to Bevacizumab
For further deciphering whether miR-199a-5p increased the sensitivity of A549 cells to Bevacizumab by suppressing the expression of HIF-1α and the activation of STAT3, miR-199a-5p mimics and HIF-1α or STAT3 overexpression plasmids were co-transfected into A549 cells (Fig. 9A-E). It was experimentally implicated that both HIF-1α and STAT3 were capable of remarkably eliminating the effects of miR-199a-5p on the sensitivity of A549 cells to Bevacizumab in vitro and in vivo (Fig. 9F-J). These data validated that miR-199a-5p was capable of sensitizing A549 cells to Bevacizumab through regulating HIF-1α and STAT3.

Discussion
Multiple miRNAs have been identi ed as potential diagnostic biomarkers and therapeutic targets for NSCLC. For example, expression of miR-128-3p was signi cantly up-regulated in NSCLC tissues by comparison with adjacent normal tissues; its overexpression remarkably induced the migration and invasion of A549, Calu-3 and H520 cells [17]. In the present research, miR-199a-5p was found to be dramatically down-regulated in NSCLC tissues, serum samples of NSCLC patients and multiple NSCLC cell lines. Gain-of-function and loss-of-function experiments further illustrated that miR-199a-5p constrained the proliferation, migration and invasion, and facilitated the apoptosis of NSCLC cells, indicative of a prospective value regarding miR-199a-5p in NSCLC therapeutics. Some other studies also demonstrate that miR-199a-5p represses the malignancy of NSCLC cell lines, such as H1299, A549 and SPCA-1 cells, which is consistent with our demonstrations [18,19]. The underexpression of miR-199a-5p in NSCLC has been reported by several previous reports [6,7,18,19]. "competitive endogenous RNA (ceRNA)" mechanism can probably contribute to the dysregulation of miR-199a-5p in NSCLC. It is reported that, LINC01123 sponges miR-199a-5p as a ceRNA to negatively regulate its expression in NSCLC cells [18]; similarly, in NSCLC cells, miR-199a-5p is reported to be adsorbed and repressed by lncRNA PVT1 [19]. Interestingly, in cardiomyocytes, pSTAT3 is able to bind to the promoter region of miR-199a-2 gene, and can repress its transcription and down-regulate its expression, which is more signi cant under hypoxic conditions, suggesting miR-199a-5p is modulated by STAT3 signaling and HIF-1α signaling [12,20]. In the present study, for the rst time, we demonstrated that overexpression of STAT3 or HIF-1α increased the expression of miR-199a-5p in NSCLC cells, which indicated that the hypoxic tumor microenvrionment and activation of STAT3 signaling contributed the dysregulation of miR-199a-5p in NSCLC cells.
HIF-1α, recognized as oxygen-labile subunit of HIF-1, contains the transactivation domains responsible for HIF-1α transcriptional activity [21,22]. Despite the fact that HIF-1α is merely highly expressed during hypoxic condition, it is also detectable in normoxic condition. Hypoxia is a common characteristic in the microenvironment of various types of solid tumors, promoting HIF-1α expression [22][23][24]. In turn, HIF-1α is conducive to the adjustment of tumors to hypoxia through transcriptional activation of more than 100 downstream genes including LEP, EPO, PKM, etc; in this regard, HIF-1α further promotes proliferation and growth of tumor cells [23]. In NSCLC, HIF-1α is up-regulated in tumor tissues and cell lines, and its overexpression facilitates the proliferation, migration and invasion of cancer cells; what's more, the 5-year survival rate of patients with low expression level of HIF-1α is higher than those with high expression level of HIF-1α [25][26][27]. For shedding more light upon the underlying mechanism by which miR-199a-5p exhibited its tumor-suppressive role in NSCLC, miRmap, miRanda and TargetScan databases were employed for prediction of target gene of miR-199a-5p in this study. Interestingly, the gene of HIF-1α, HIF1A, was validated as a target gene of miR-199a-5p in NSCLC in the present study. Our data suggested that the dysregulation of HIF-1α in NSCLC is not only due to the hypoxic microenvrionment, but also partly resulted from the down-regulation of upstream miRNAs.
STAT3, a member of signal transducer and activator of transcription (STAT) family, regulates the expressions of genes relevant to cell cycle, cell survival, and immune response. In recent years, STAT3 has been found to be constitutively activated in multiple types of human cancers, indicating that STAT3 is a valuable target for cancer therapy [28]. STAT3 has been identi ed to be abnormally increased in NSCLC tissues and cell lines, and knockdown of STAT3 can induce apoptosis, reduce proliferation, migration and invasion of A549 and H1975 cells [29]. Consistently, we demonstrated that transfection of STAT3 overexpression plasmids notably induced proliferation, migration and invasion of NSCLC cells. It is noteworthy that HIF-1α is previously reported to activate STAT3 by means of repressing miR-34a in colorectal cancer [11]. Furthermore, in head and neck squamous cell carcinoma, with static inhibition of STAT3 activation, expression in HIF-1α was repressed [30]. In the present work, we demonstrated that HIF-1α overexpression activated STAT3, and STAT3 overexpression repressed the expression of miR-199a-5p, but promoted the expression of HIF-1α. Altogether, our study presented a novel positive feed-back loop, which was formed by miR-199a-5p, HIF-1α and STAT3 in NSCLC, and this positive feed-back loop is crucial to clarify the mechanism of hyperactivation of HIF-1α pathway and STAT3 signaling in NSCLC tissues.
At the last part of our present study, we analyzed the sensitivity of A549 cells to Bevacizumab and found the positive feed-back loop also contributed to Bevacizumab sensitivity. Bevacizumab, possessing high e cacy and safety, is a monoclonal anti-VEGF antibody and an encouraging target drug for NSCLC, particularly advanced NSCLC patients [31,32]. There remains little study investigating the molecular mechanism regarding Bevacizumab sensitivity in NSCLC at the present. We found the positive feed-back loop miR-199a-5p-HIF-1α-STAT3 also exists after Bevacizumab treatment and miR-199a-5p enhanced sensitivity of A549 cells to Bevacizumab. Our study provides a new thought to further improve outcomes for Bevacizumab therapy. miR-199a-5p may have the potential to improve prognosis for NSCLC patients in advanced stages.
The loop contributes to NSCLC progression and resistance to Bevacizumab. The results provide important information regarding molecular regulating networks in NSCLC.

Declarations
Ethics approval and consent to participate

Consent for publication
Not applicable.

Availability of data and material
The data used to support the ndings of this study are available from the corresponding author upon request. Figure 1 A positive feed-back loop among miR-199a-5p, HIF-1α and STAT3 Figure 2 Expression of miRNAs in GSE135981 The heatmap and volcano plot were established with data in GSE135981. (A) MiRNAs with signi cant changes of expression level between tumor tissues versus normal lung tissues and |log2FC| > 1.5 were shown in heatmap. (B) All the miRNAs were shown in volcano plot and miRNAs signi cantly upregulated with log2FC > 1.5 were painted red, while miRNAs signi cantly downregulated with log2FC < -1.5 were painted green.  cells. *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001, respectively. HIF-1α and miR-199a-5p in NSCLC tissues. *** represents p < 0.001.